The present invention relates to human and mouse Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger (sodium ion-driven chloride/bicarbonate exchanger) proteins, which are a class of proteins involved in intracellular pH regulation. More specifically, the present invention relates to sodium ion-driven chloride/bicarbonate exchanger proteins, cells designed to express one of the proteins, which cells are of a species different from the origin of the one of the proteins expressed, DNAs encoding the proteins, antibodies to the proteins, and a method for selecting agonists/antagonists of the sodium ion-driven chloride/bicarbonate exchanger proteins.
Regulation of intracellular pH (pHi) in response to various stimuli is a critical one among a number of cellular functions. A family of bicarbonate transporters is a major pHi regulator under physiological conditions in animal cells. Bicarbonate (HCO3xe2x80x94) transporters are divided into four groups according to their functions [Boron, W. F. et al., J. Exp. Biol., 200:263-268(1997)]: Na+-independent Clxe2x80x94/HCO3xe2x80x94 exchanger (alternatively called an anion exchanger, AE), Na+xe2x80x94HCO3xe2x80x94 cotransporter (NBC), K+xe2x80x94HCO3xe2x80x94 cotransporter, and Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger. Three AEs and three NBCs have been cloned and functionally characterized, but the molecular structure of the K+xe2x80x94HCO3xe2x80x94 cotransporter and the Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger have remained unknown.
A Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger was first discovered in invertebrate neurons and was later found in vertebrate neurons as well as non-neuronal cells, including brain, vascular endothelial cells, sperm, kidney and pancreatic xcex2-cells. Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger is an intracellular pH regulator that transports extracellular Na+and HCO3xe2x80x94 into the cells in exchange for intracellular Clxe2x80x94, thereby playing an important role in cellular alkalinization.
In pancreatic xcex2-cells, glucose is the most important physiological regulator of insulin secretion. Glucose metabolism induces an increase in intracellular pH in the pancreatic cells. It has been shown that this glucose-induced pHi rise is evoked primarily by the action of Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger [Pace, C. S. et al., J. Membrane Biol., 73:39-43(1983)].
Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger is thus an important intracellular pH regulator in various cells, but its molecular basis is not known. Analysis of the molecular structure and function of Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger should be valuable not only for functional analysis of insulin secretion by pancreatic xcex2-cells but also for screening as well as for drug designing based on its molecular structure aimed at the development of therapeutics of diabetes mellitus.
On the above background, the present invention has as its objective to clone Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchangers, thereby obtaining their DNA for sequencing, providing cells of a different species expressing the DNAs, and determining the structure and function of the Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchangers.
Thus, the present invention provides a Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger protein comprising the amino acid sequence set forth as SEQ ID NO:2 or NO:4 in the Sequence Listing.
The present invention further provides a protein comprising an amino acid sequence having deletion, substitution, addition or insertion of one or more amino acids relative to the amino acid sequence set forth as SEQ ID NO:2 or NO:4 in the Sequence Listing and which, when expressed in a cell, functions as Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger.
The present invention further provides an above protein wherein the Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger, dependently upon both of extracellular bicarbonate and intracellular chloride ions, takes up extracellular sodium ion into the cell and transport intracellular sodium ion out of the cell.
The present invention further provides a cell in which one of the above proteins is expressed, wherein the cell is of a species different from the species of origin of the one of the proteins. Non-limiting examples of such cells of different species include Xenopus laevis oocytes and HEK293 cells. Expression of a Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger in such cells of different species may be achieved by transfection of a DNA encoding the Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger or by introduction of a cRNA corresponding to the Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger.
The present invention further provides antibodies to the above proteins. The antibodies may be monoclonal or polyclonal.
The present invention further provides a method for selection of agonists and antagonists of Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger, which method comprises bringing a cell of a different species expressing the protein into contact with a candidate compound, measuring the function of the Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger, comparing the result thus obtained with a result obtained by measuring the function of the sodium ion-driven chloride/bicarbonate exchanger of the cell which has not been brought into contact with the candidate compound, and thereby determining whether or not the candidate compound enhances or inhibits the function.
The present invention further provides a DNA comprising the nucleotide sequence set forth as SEQ ID NO:1 or NO:3 in the Sequence Listing, a DNA  greater than 2 comprising a nucleotide sequence consisting of nucleotides 67 through 3330 in the nucleotide sequence set forth as SEQ ID NO:1 in the Sequence Listing, and a DNA comprising a nucleotide sequence consisting of the nucleotides 83 through 3346 in the nucleotide sequence set forth as SEQ ID NO:3 in the Sequence Listing.
The present invention further provides a DNA comprising a nucleotide sequence having deletion, substitution, addition or insertion of one or more nucleotides relative to a DNA comprising a nucleotide sequence consisting of the nucleotides 67 through 3330 in the nucleotide sequence set forth as SEQ ID NO:1 in the Sequence Listing, and encoding:
(1) a protein comprising the amino acid sequence set forth as SEQ ID NO:2 in the Sequence Listing, or
(2) a protein comprising an amino acid sequence having deletion, substitution, addition or insertion of one or more amino acids relative to the amino acid sequence set forth as SEQ ID NO:2 in the Sequence Listing, which protein, when expressed in a cell, functions as Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger.
The present invention still further provides a DNA comprising a nucleotide sequence having deletion, substitution, addition or insertion of one or more nucleotides relative to a DNA comprising a nucleotide sequence consisting of the nucleotides 83 through 3346 in the nucleotide sequence set forth as SEQ ID NO:3 in the Sequence Listing, and encoding:
(1) a protein comprising the amino acid sequence set forth as SEQ ID NO:4 in the Sequence Listing, or
(2) a protein comprising an amino acid sequence having deletion, substitution, addition or insertion of one or more amino acids relative to the amino acid sequence set forth as SEQ ID NO:4 in the Sequence Listing, which protein, when expressed in a cell, functions as Na+-driven Clxe2x80x94/HCO3xe2x80x94 exchanger.